Detection of antibodies to squalene in serum

ABSTRACT

The invention is a method for detecting squalene in sera.

This application is a divisional of application Ser. No. 09/859,389filed May 18, 2001 now U.S. Pat. No. 6,900,025, which claims the benefitof Provisional Application Ser. No. 60/205,041 filed May 18, 2000.

I. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Not applicable.

II. FIELD OF THE INVENTION

Squalene (SQE) is a triterpenoid hydrocarbon oil, C₃₀H₅₀, that is widelyproduced by both plants and animals, and is present in human food. SQUis also widely used in skin cosmetics. In humans, SQE serves a asprecursor in the synthesis of cholesterol and all of the steroidhormones (Mayes, 1996; Granner, 1996) (FIG. 1). Both SQE and cholesterolare transported in the blood on very low density lipoproteins (VLDL) andlow density lipoproteins (LDL) (Miettinen, 1982; Koivisto and Miettinen,1988). Squalene and cholesterol are also synthesized in the liver and inthe epidermis of the skin where SQE comprises a large amount of the oilsecreted by sebaceous glands (Stewart, 1992). Because it is anaturally-occurring biodegradable oil, SQE and its hydrogenatedderivative squalane (SQA) have each been proposed for use as the oilcomponent of oil-in-water (o/w) emulsions for new generations ofadjuvants for vaccines (Minutello et al., 1999).

Immunization against a potential antigen such as SQE presents aparticular Catch-22 challenge: first, there have never been any previousantibodies developed that could serve as validated positive controls foranti-SQE antibodies, and second, there is no validated assay availablefor detecting antibodies to SQE. To overcome this difficult dilemma inthe present study, the horns of which are the simultaneous lack ofpositive antibody controls from immunized animals and lack of avalidated assay for antibodies to SQE, our first goal was to inject SQEinto mice to try to create antibodies that could potentially bevalidated as having anti-SQE activity. The second goal, namely thecreation of monoclonal antibodies that could serve as positive antibodycontrols, was considered to be a requirement in the ultimate third goalof development of a valid immunoassay for detection of specificantibodies to SQE.

It has been previously reported that SQE incorporated intonon-phospholipid liposomes has an adjuvant effect on the induction ofantibodies to a non-phospholipid liposomal protein, but the adjuvanteffect was not enhanced further by simultaneous incorporation of lipid A(Gupta et al., 1996). Although incorporation of lipid A without SQE intonon-phospholipid liposomes was not tested in the latter study, thepotent adjuvant effect of liposomal SQE for liposomal protein wasclearly shown. This adjuvant effect of liposomal SQE therefore may alsohave played a role in our liposomes in the induction of antibodies toSQE.

As with 71% cholesterol in liposomes, the biophysical conformation of71% SQE in our liposomes is not completely clear. Previous work hassuggested that SQE locates itself in the most disordered region ofliposomes, predominately in the center area of the liposomal bilayer(Lohner et al., 1993). Because of this it has been proposed that SQEadopts a coil rather than an extended conformation when it is located inthe bilayer interior. Although relatively small amounts of SQE have adisruptive effect on the liposomal bilayer and lead to formation oftubules having the H_(II) conformation in liposomes containingphosphatidylethanolamine (Lohner et al., 1993), the H_(II) conformationdoes not occur in liposomes, such as ours, that lackphosphatidylethanolamine. Nonetheless, the reported ability of SQE tolower the transition temperature of phosphatidylcholine and to causedisruption in the stability of the liposomal bilayer (Lohner et al.,1993), together with the high concentrations of SQE combined with lipidA in the liposomes used in this study, may play a role in the potentability of these liposomes to induce antibodies to SQE.

From a purely structural standpoint, it may not be initially surprisingthat antibodies to SQE can be induced in a similar manner to thoseagainst cholesterol, in view of the striking apparent structuralsimilarity of SQE and cholesterol (FIG. 1). Balanced against this,however, is the observation that the immunogenic epitope ofliposome-associated cholesterol is the polar 3-β-hydroxy group in the Aring (Dijkstra et al, 1996), and the fact that SQE not only lacks anyclosed ring, but is an exceedingly hydrophobic alkene that completelylacks any polar group.

What, if any, are the potential consequences of induction of antibodiesto SQE? A recent publication claims to have detected antibodies to SQEin sick but not in healthy individuals (Asa et al., 2000). However, webelieve that such a conclusion may be premature, based on a technicalcritique of the reported Western blot-type assay that was used (Alvingand Grabenstein, 2000). Turning again to cholesterol for comparison,SQE, as a precursor in the synthesis of cholesterol, is found nearlyeverywhere that cholesterol is found, with the apparent exception thatSQE probably does not have a structural role in promoting the stabilityof membranes. As with cholesterol, SQE circulates in the blood as aconstituent of LDL and VLDL (Miettinen, 1982; Koivisto and Miettinen,1988). Naturally-occurring antibodies to cholesterol have beendemonstrated to be present in virtually all human serum samples tested,and they have been proposed to have a vital beneficial role in thenormal regulation of LDL and VLDL metabolism (Alving and Wassef, 1999).

III. BACKGROUND OF THE INVENTION

Antibodies to SQE have had great interest in the popular press. Asa etal., described antibodies to SQE in the serum of sick Gulf War Veteransand purported that these antibodies were responsible for their disease[Asa, et al., “Antibodies to Squalene in Gulf War Syndrome,” Exp. Mol.Path. 68:55 (2000)]. Asa's assay has been criticized for technicalreasons, which render the reported results as highly questionable orinvalid [Alving, et al., Letter to the Editor. Exp. Mol. Path. 68:196(2000)]. U.S. Pat. No. 6,214,566 (Asa, et al.) discloses an immunoassayfor detecting anti-squalene antibodies.

In order to develop a highly reliable assay for antibodies to SQE, wedeveloped murine monoclonal antibodies to SQE to serve as positivecontrols [Matyas, et al., “Induction and detection of antibodies tosqualene,” J. Immunol. Meth. 245:1 (2000)]. These monoclonal were usedto develop an assay for measuring antibodies to squalene in human serum.

IV. SUMMARY OF THE INVENTION

In view of the success that was previously found using lipid A as anadjuvant for inducing antibodies to cholesterol (Swartz et al., 1988;Alving and Swartz, 1991), immunization strategies using SQE combinedwith lipid A were employed in attempting to induce antibodies to SQE.The results demonstrate that murine antibodies to SQE can be induced byinjection of SQE-loaded liposomes containing lipid A, and the antibodiescan be detected by an ELISA in which the antigen is coated onhydrophobic membranes instead of polystyrene microtiter wells. This hasallowed creation of an immunoassay for demonstrating that mAbs to SQEcan be produced that differentiate SQE from SQA.

The assay used in the development of the monoclonal antibodies to SQEused 96 well plates containing PVDF membranes. This assay was highlyreproducible, but was very labor intensive; limiting the number ofsamples that could be assayed. Studies with different lots of platesrevealed variations in assay results (data not shown). In addition, theassay used PBS-4% fetal bovine serum (FBS) as a blocker/dilutent buffer.Like human serum, FBS undoubtedly contains SQE. This serum SQE couldpotentially compete for antibody binding in the assay. In order toovercome these shortcomings, we describe a modified, highly reproducibleassay for antibodies to SQE.

A new highly reproducible and high through-put assay for measuringantibodies to SQE was developed. The assay utilizes Costar 96 well Ubottom sterile tissue culture plates, which are not routinely used forELISA assay. None of the standard ELISA plates tested were useful forthis assay; most gave high background.

The accompanying drawings show illustrative embodiments of the inventionfrom which these and other of the objectives, novel features andadvantages will be readily apparent.

V. DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of squalene and cholesterol.

FIG. 2 shows the binding activity of mouse serum IgM to SQE by ELISA.Mice were immunized biweekly with: A) Liposomes containing lipid A as anadjuvant and composed of DMPC/DMPG/SQE in a molar ratio of 9:1:2.5(group 4); or B) an emulsion containing of 20% SQE, 5% Tween 80, 5% Span85 and lipid A (group 6); or C) the above liposomes containing lipid Aas an additional adjuvant. Serum obtained from these mice were tested byELISA as described in the Materials and Methods. Polystyrene “U” bottomplates were coated with 10 μg/well of SQE in ethanol. Binding activityof the indicated dilutions of preimmune and immune serum was assayed atthe indicated time points. Results are presented as the mean absorbancefrom triplicate wells containing squalene subtracted from the absorbanceof triplicate wells lacking squalene±SD.

FIG. 3 shows the end point dilution IgM titers of immune mouse serumagainst SQE, and liposomes containing or lacking SQE. Serum from miceimmunized biweekly with liposomes containing lipid A as an additionaladjuvant and composed of DMPC/DMPG/SQE in a molar ratio of 9:1:2.5(group 4) were tested by ELISA. Capture antigens for the assay consistedof SQE or of liposomes containing or lacking squalene. Polystyrene “U”bottom plates were coated with 10 μg/well of SQE in ethanol, or with theequivalent amount of L(SQE), or with the equivalent amount of L. Theresults shown were obtained by subtracting the absorbance of triplicatewells containing the appropriate capture antigen from the absorbance oftriplicate wells lacking antigens. Endpoint IgM antibody titers werecalculated from the highest dilution of serum giving twice theabsorbance of the background.

FIG. 4 shows the comparative binding of a mAb to SQE or SQA coated onPVDF or PS flat bottom plates. Each well contained 10 μg of SQA or SQEdissolved in 0.1 ml of isopropanol, or isopropanol alone (control), asappropriate, at the concentrations indicated. The culture supernatant ofa mAb was diluted in PBS-4% fetal bovine serum (PVDF plates) or PBS-0.3%gelatin (PS plates). ELISAs were performed as described in the Methodssection for the PVDF and PS plates, respectively. Similar results wereobserved with 8 other clones. Values are the mean±standard deviation oftriplicate wells.

FIG. 5 shows the specific binding of mAb clone 5 to SQE, but not SQA.The assay was conducted with PVDF plates as described in the legend toFIG. 4.

FIG. 6 shows the binding of mAb clone 15 to SQE and cross-reactivitywith SQA. The assay was conducted with PVDF plates as described in thelegend to FIG. 4.

FIG. 7 shows the reactivity of monoclonal antibodies to liposomescontaining or lacking SQE or SQA. L(SQE), L(SQA) and L (33 nmol ofphospholipid) in 0.05 ml of PBS was placed in each well of a PS“U”bottom plate. The plates were processed as described in the Methods.Culture supernatants from the indicated clones were diluted in PBS-0.3%gelatin and 0.05 ml was placed in each well. Values are themean±standard deviation of triplicate wells. A, B, C, D: Binding of theindicated culture supernatants to L(SQE), L(SQA), and L. E: Negativecontrols consisting of binding of an irrelevant IgM secreting clone 2D4(IgM anti-G_(M2)).

FIG. 8 shows the binding of antiserum IgM to SQE and SQA on PVDF and PSflat bottom ELISA plates. Pre-immune or 3 day post-immune serum frommice immunized with liposomes containing 71% SQE was diluted in PBS-4%fetal bovine serum (PVDF plates) or PBS-0.3% gelatin (PS plates). ELISAswere performed as described in the Methods section for the PVDF and PSplates, respectively. Values are the mean±standard deviation oftriplicate wells.

FIG. 9 shows the comparison of plates from different manufacturers withPBS-4% FBS as a blocker/diluent. Plates were coated with 100 nmol ofSQE. mAbs SQE #16 and SQE #18 were diluted 1:10. The normal mouse serumand anti-SQE serum were diluted 1:50. The ELISA was performed asdescribed for the standard protocol using PBS-4% FBS. Values are themean of triplicate determination±standard deviation.

FIG. 10 shows the comparison of different blocker/diluents on thebinding of antibodies to SQE. Costar U bottom plates were used. Plateswere coated with 100 nmol of SQE. Clone SQE #16 (A) and SQE #18 (B) werediluted 1:10 in PBS, pH 7.4 containing the blocker/diluents indicated.Normal mouse serum (C) and anti-SQE serum (D) were diluted 1:50. TheELISA was performed as described for the standard protocol. Values arethe mean of triplicate determination±standard deviation.

FIG. 11 shows the comparison of different amounts of BSA used as theblocker/diluent. Costar U bottom plates were used. Plates were coatedwith 100 nmol of SQE. Antibodies were diluted in PBS containing thepercent BSA indicated. Clone SQE #16 (A) and SQE #18 (B) were diluted1:10. Normal mouse serum (C) and anti-SQE serum (D) were diluted 1:50.The ELISA was performed as described for the standard protocol. Valuesare the mean of triplicate determination±standard deviation.

FIG. 12 shows the binding of anti-SQE antibodies to SQE-coated plates asa function of incubation temperature. Plates were coated with 100 nmolof SQE. PBS-2% BSA was used as a blocker/diluent, which was equilibratedto temperature indicated. Clone SQE #16 (A) and the anti-SQE serum (C)were diluted 1:50 and 1:100 respectively. The ELISA was performed asdescribed for the standard assay except the incubations were at thetemperature indicated. Values are the mean of triplicatedetermination±standard deviation.

FIG. 13 shows the binding of anti-SQE antibodies to SQE-coated plates asa function of primary antibody incubation time. Plates were coated with100 nmol of SQE. PBS-2% BSA was used as a blocker/diluent. Clone SQE #16and the anti-SQE serum were diluted 1:100. The ELISA was performed asdescribed for the standard protocol except for the primary antibodyincubation time. Values are the mean of triplicatedetermination±standard deviation.

FIG. 14 shows the effect of secondary antibody incubation time on ELISAabsorbance of antibodies binding to SQE. Plates were coated with 100nmol of SQE. PBS-2% BSA was used as a blocker/diluent. Clone SQE #16 andthe anti-SQE serum were diluted 1:40 and 1:50, respectively. The ELISAwas performed as described for the standard protocol except for thesecondary antibody incubation time. Values are the mean of triplicatedetermination±standard deviation.

FIG. 15 shows the binding of anti-SQE antibodies as a function ofSQE-coated on the well. Plates were coated with the amount of SQEindicated. PBS-2% BSA was used as a blocker/diluent. Clones SQE #14 (A)and SQE #16 (B) and serum (C) was diluted as indicated. The ELISA wasperformed as described for the standard protocol. Values are the mean oftriplicate determination±standard deviation.

FIG. 16 shows the comparison of different lots of plates. Plates werecoated with 10 nmol of SQE. PBS-2% BSA was used as a blocker/diluent.Clone SQE #14 (A), clone SQE #16 (B) anti-SQE serum (C) were used asprimary antibodies. The ELISA was performed as described for thestandard protocol. Each symbol is a plate with a different lot number.Values are the mean of triplicate determination±standard deviation.

FIG. 17 shows the day to day reproducibility of the ELISA assay forantibodies to SQE. Plates were coated with 10 nmol of SQE. PBS-2% BSAwas used as a blocker/diluent. Experiments 1 and 2 were done on separatedays using the standard protocol. Values are the mean of triplicatedetermination±standard deviation.

FIGS. 18-22 shows the results of the experiments described in Example19, illustrating the applicability of the method of the presentinvention to detecting anti-squalene antibodies in human sera.

VI. DETAILED DESCRIPTION OF THE INVENTION

The present invention is monoclonal antibody that specifically binds tosqualene, and methods for producing the monoclonal antibody. Amonoclonal antibody that specifically binds to squalene has beendeposited in the American Type Culture Collection, and has receivedAccession No. PTA 6538 and PTA 6539. “Squalene” refers to a hydrocarbonof the chemical formula C₃₀H₅₀[2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene], CASNumber [111-02-4].

The present invention is also the use of that monoclonal antibody, or asegment or portion thereof, in an immunoassay for the detection ofanti-squalene antibodies. “Anti-squalene antibody” refers to an antibodycapable of complexing with squalene. Such an antibody may complex withsqualene, or with any antigenic epitope presented by squalene.

The present invention is also directed to an immunoassay for detectinganti-squalene antibodies. In preferred embodiments of the invention, theimmunoassay is specific for anti-squalene antibodies. In most preferredembodiments of the invention, the immunoassay is capable ofdifferentiating between anti-squalene antibodies and anti-squalaneantibodies.

The test sample may generally be any type of biological materialcontaining antibodies. Such materials may be processed so that they areprovided in a suitable form. The test sample is preferably provided froma bodily fluid, more preferably is provided from blood, and mostpreferably provided from serum. The organism providing the test samplemay generally be any organism which contains antibodies. The organismpreferably is a mammal, and more preferably is a human.

In accordance with some embodiments of the present invention, theimmunoassay uses a polystyrene support in combination with certainblockers/diluents. In accordance with the present invention, fetalbovine serum should not be used as a blocker/diluent because it appearsto compete with antibody binding in the assay. Not intending to belimited to a particular theory, it is believed that fetal bovine serumitself includes an amount of squalene sufficient to block or diminishantibody binding.

In preferred embodiments of the invention, the assay uses ablocker/diluent that does not compete with squalene and/or anti-squaleneantibodies. Exemplary blockers/diluents suitable for use withpolystyrene supports include but are not limited to phosphate bufferedsaline (PBS), bovine serum albumin (BSA), gelatin, casein, orcombinations or mixtures thereof. Preferred blockers/diluents includeBSA; most preferred blockers/diluents include BSA and PBS. As noted inmore detail in the Examples, the preferred amount of BSA is up to about5% by volume BSA, for example, from about 1% to about 2%.

In accordance with some embodiments of the present invention, theimmunoassay may use a hydrophobic membrane support, preferablypolyvinylidene difluoride.

In accordance with the present invention, any assay suitable for usewith a monoclonal antibody or antibody fragment may be used to detectsqualene antibodies. Preferred assays are a radioimmunoassay and ELISA.

The present invention also includes preparing a monoclonal antibody thatspecifically binds to or reacts with squalene. In preferred embodimentsof the invention, the monoclonal antibody binds to squalene but notsqualane (hydrogenated form of squalene).

The present invention also includes a method for detecting anti-squaleneantibodies. The present invention also includes a method for selectivelydetecting anti-squalene antibodies, i.e., differentiating betweenanti-squalene antibodies and anti-squalane antibodies.

The present invention also includes a kit for detecting anti-squaleneantibodies, said kit including one or more of the following: componentsused for a radioimmunoassay; components used for ELISA; one or moremonoclonal antibodies; one or more antibody fragments; one or morewashes; one or more buffers; one or more detection agents or labels,including but not limited to peroxidase; and one or more solid supportsconfigured and suitable for use with the particular assay beingconducted. A diagnostic kit may be designed to aid the performance ofthe above method. Such a kit may contain vessels containing squalene andthe indicator regent, respectively.

Exemplary solid supports include but are not limited to polystyrene orpolyvinyldiene fluoride (PVDF).

Each of these elements will now be described in more detail.

Exemplary binding agents include, but are not limited to: monoclonalantibodies (“MAb”); chimeric monoclonal antibodies (“C-MAb”); humanizedantibodies; genetically engineered monoclonal antibodies (“G-MAb”);fragments of monoclonal antibodies (including but not limited to“F(Ab)₂”, “F(Ab)” and “Dab”); single chains representing the reactiveportion of monoclonal antibodies (“SC-MAb”); antigen-binding peptides;tumor-binding peptides; a protein, including receptor proteins; peptide;polypeptide; glycoprotein; lipoprotein, or the like, e.g., growthfactors; lymphokines and cytokines; enzymes, immune modulators;hormones, for example, somatostatin; any of the above joined to amolecule that mediates an effector function; and mimics or fragments ofany of the above. The binding agent may be labeled or unlabeled.

A binding agent according to the invention is preferably a monoclonal orpolyclonal antibody. The antibody includes, but is not limited to nativeor naked antibodies; modified antibodies, such as activated orphotoactivated antibodies. As used herein, native refers to a natural ornormal antibody; naked refers to removing a non-native moiety, e.g.,removing the label from a labeled antibody.

Methods for producing and obtaining an antibody are well known by thoseskilled in the art. An exemplary method includes immunizing any animalcapable of mounting a usable immune response to the antigen, such as amouse, rat, goat sheep, rabbit or other suitable experimental animal. Inthe case of a monoclonal antibody, antibody producing cells of theimmunized animal may be fused with “immortal” or “immortalized” human oranimal cells to obtain a hybridoma which produces the antibody. Ifdesired, the genes encoding one or more of the immunoglobulin chains maybe cloned so that the antibody may be produced in different host cells,and if desired, the genes may be mutated so as to alter the sequence andhence the immunological characteristics of the antibody produced.Fragments of binding agents, may be obtained by conventional techniques,such as by proteolytic digestion of the binding agent using pepsin,papain, or the like; or by recombinant DNA techniques in which DNAencoding the desired fragment is cloned and expressed in a variety ofhosts. Irradiating any of the foregoing entities, e.g., by ultravioletlight will enhance the immune response to a multi-epitopic antigen undersimilar conditions. Various binding agents, antibodies, antigens, andmethods for preparing, isolating, and using the binding agents aredescribed in U.S. Pat. No. 4,471,057 (Koprowski), U.S. Pat. No.5,075,218 (Jette, et al.), U.S. Pat. No. 5,506,343 (Fufe), and U.S. Pat.No. 5,683,674 (Taylor-Papadimitriou, et al), all incorporated herein byreference. Furthermore, many of these antibodies are commerciallyavailable from Centocor, Abbott Laboratories, Commissariat a L'EnergieAtomique, Hoffman-LaRoche, Inc., Sorin Biomedica, and FujiRebio.

The compositions and methods of the present invention are suitable foruse in any immunoassay capable of detecting an antibody or the likebound to an antigen. Exemplary assays include labeled binding reagentassays, including noncompetitive and competitive binding assays,including assays in which the solid phase is the binding reagent or theligand, and sandwich assays, including precipitation, radioimmunoassay,or enzyme-linked immunosorbent assay. It is intended that the inventionis not to be limited by the type of immunoassay employed or the specificprotocol used in performing the assay. Exemplary immunoassays techniquesare shown in the Examples.

The squalene provided in the above method may be immobilized on a solidsupport. The solid support may be provided in one of many differentforms. These forms may include a membrane, filter, plastic, bead,agarose bead, SEPHAROSE (SEPHAROSE is a registered trademark ofPharmacia Biotech, Piscataway, N.J.) Bead, or magnetic bead.

In addition to the different forms, the solid support may be made from avariety of materials. The solid support is preferably nitrocellulose,polyvinylidene difluoride, nylon, rayon, cellulose acetate, agarose,SEPHAROSE, metal, polypropylene, polyethylene, polystyrene, polyvinylchloride, polyvinyl acetate, polyamide, polyimide, polycarbonate,polyether, polyester, polysulfono, polyacetal, or polymethylmethacrylate, more preferably is polypropylene, polystyrene,polyvinylchloride, polyamide, polycarbonate, polyether, polymethylmethacrylate, nitrocellulose, polyvinylidene difluoride, or nylon, andmost preferably is nitrocellulose.

The squalene may generally be from any source. Commercial preparationsare readily available (Sigma, St. Louis, Mo.). Alternatively, it may besynthesized from various precursors or obtained from an organism.Squalene is a relatively large hydrocarbon which may contain multipleantigenic epitopes. As a result any portion of squalene containing andantigenic epitope may be used in place of squalene in the presentinvention.

It is preferred that during the assay process, substantially all of atleast one predetermined ligand or ligand receptor remains in apredetermined position. Any technique for immobilizing a ligand orligand receptor is included in the scope of the present invention. In apreferred embodiment, a ligand or ligand receptor is bound orimmobilized on or in a solid phase. Typical immobilization mechanismsinclude, but are not limited to, covalent binding, non-covalent binding,chemical coupling, physical entrapment, and adsorption.

Included within the scope of the present invention is changing orincorporating different surface properties on the membrane in order toachieve a desired result, e.g., the surface properties of a membranedesigned for a competitive binding assay for a hormone may be differentthan an immunometric assay for a therapeutic drug. For example, it hasbeen shown that treating the surface of a hydrophilized PVDF membranewith ethanolamine reduces the non-specific binding of the membranesurface. Selection of a particular surface treatment agent or surfaceproperty may be based on the desired chemical characteristic to beimparted to the surface; the inability or reduced capability ofdenaturing or impairing the functionality of a bioactive agent on or inthe reaction zone; the desire to effect a certain orientation of animmobilized bioactive molecule; the desire to promote long-termstability of an immobilized bioactive molecule; the inclusion of adesired nucleophilic substituent; and the availability and cost oftreatment agents. The use of other surface treatment agents, includingbi-functional or multi-functional reagents, to affect the surfaceproperties of the membrane are included within the present invention.

There are many other suitable detection methods compatible with theinstant invention. In each case, the detection agent and its method ofuse are well known to one of ordinary skill in the art. The indicatorreagent is typically conjugated to a detectable label. The detectablelabel may be an enzyme, such as alkaline phosphatase, β-galactosidase,or peroxidase; a protein, such as biotin or digoxin; a fluorochrome,such as rhodamine, phycoerythrin, or fluourescein; a fluorescentprotein, such as GFP or one of its many modified forms; a radioisotope;or a nucleic acid segment. Enzymes, such as horseradish peroxidase,alkaline phosphatase, and β-galactosidase, may also be used asdetectable labels. Detection agents for enzymes generally utilize a formof the enzyme's substrate. The substrate is typically modified, orprovided under a set of conditions, such that a chemiluminescent,colorimetric, or fluorescent signal is observed after the enzyme andsubstrate have been contacted (Vargas, et al. Anal Biochem 209: 323,1993).

A signal-producing agent refers to any agent or marker which produces adetectable signal or which permits the detection of a ligand orligand-receptor. Preferred signal-producing agents are those whichpermit detection of the analyte without instruments, preferably byvisual means. Exemplary signal-producing agents include, but are notlimited to color forming agents, such as an enzyme, polymer containingdyes, chemiluminescent agents, fluorescent agents, radioisotopes orferromagnetic particles. The color forming agent may be a coloredparticle, a colored molecule or some species, such as an enzyme, whichis capable of triggering a sequence of events leading to the formationof a colored marker. The colored molecule may be a fluorescent dye, suchas fluorescein or rhodamine; a chemiluminescent compound; abioluminescent compound; or a compound that may be detected by theabsorption of electromagnetic radiation (and possible reemission ofradiation at another wavelength), including ultraviolet radiation,visible radiation and infrared radiation. The colored molecule may bedirectly or indirectly conjugated to a ligand or ligand-receptor.Alternatively, the colored molecule may be incorporated in a particle,particularly a microsome.

Enzymes, useful as color forming agents, include alkaline phosphatase,horseradish peroxidase or B-galactosidase. Such enzymes are often usedin conjunction with a chromogenic substrate.

I the methods of the present invention, it may be desirable to controlor specify the amount of squalene bound to the solid support.Determining the appropriate amount of squalene for a particular assay iswell within the capability of one skilled in the art. The presentinventors have found that, for the murine assays shown in the Examples,up to about 100 nmol, preferably between about 7.5 and about 100 nmol,and most preferably, between about 10 and about 25 nmol of squaleneyields a reproducible assay. For the human assays shown in the Examples,up to about 500 nmol, preferably between about 7.5 and about 100 nmol,and most preferably, between about 7.5 and about 20 nmol of squaleneyields a reproducible assay.

REFERENCES

-   Alving, C. R. 1986. Antibodies to liposomes, phospholipids, and    phosphate esters. Chem. Phys. Lipids 40, 303.-   Alving, C. R., Grabenstein, J. 2000. Letter to the editor. Exp. Mol.    Path. (in press).-   Alving, C. R., Swartz, Jr., G. M. 1991. Antibodies to cholesterol,    cholesterol conjugates, and liposomes: Implications for    atherosclerosis and autoimmunity. Crit. Rev. Immunol. 10, 441.-   Alving, C. R. and Wassef, N. M. 1999. Naturally-occurring antibodies    to cholesterol: a new theory of LDL cholesterol metabolism.    Immunology Today 20, 362.-   Alving, C. R., Shichijo, S., Mattsby-Baltzer, I., Richards, R. L.,    Wassef, N. M. 1993. Preparation and use of liposomes in    immunological studies. In: G. Gregoriadis (Ed.) Liposome Technology,    vol. 3, (Second Edition), CRC Press, Inc., Boca Raton, p. 317.-   Alving, C. R., Swartz, Jr., G. M., Wassef, N. M. 1989.    Naturally-occurring autoantibodies to cholesterol in humans.    Biochem. Soc. Trans. 17 637.-   Alving, C. R., Swartz, Jr. G. M., Wassef, N. M., Ribas, J. L.,    Herderick, E. E., Virmani, R., Kolodgie, F. D., Matyas, G. R.,    Cornhill, J. F. 1996. Immunization with cholesterol-rich liposomes    induces anti-cholesterol antibodies and reduces diet-induced    hypercholesterolemia and plaque formation. J. Lab. Clin. Med. 127,    40.-   Alving, C. R., Wassef, N. M., Potter, M. 1996. Antibodies to    cholesterol: biological implications of antibodies to lipids. Curr.    Topics Microbiol. Immunol. 210, 181.-   Asa, P., Cao, Y., Garry, R. F. Antibodies to squalene in gulf war    syndrome. 2000. Exp. Mol. Path. 68, 55.-   Aniagolu, J., Swartz, Jr., G. M., Dijkstra, J., Madsen, J. W.,    Raney, J. J., Green, S. J. 1995. Analysis of anticholesterol    antibodies using hydrophobic membranes. J. Immunol. Meth. 182, 85.-   Banerji, B., Alving, C. R. 1981. Anti-liposome antibodies induced by    lipid A. I. Influences of ceramide, glycosphingolipids, and    phosphocholine on complement damage. J. Immunol. 126, 1080.-   Dijkstra, J., Swartz, Jr., G. M, Raney, J. J., Aniagolu, J., Toro,    L., Nacy, C. A., Green, S. J. 1996. Interaction of anti-cholesterol    antibodies with human lipoproteins. J. Immunol. 157, 2006.-   Granner, D. K. 1996. Hormones of the adrenal cortex. In: R. K.    Murray, D. K. Granner, P. A. Mayes and V. W. Rodwell (Eds.) Harper's    Biochemistry, 24^(th) Edition, Appleton & Lang, Stamsord, p. 547.-   Galfré, G., Milstein, C. 1981. Monoclonal antibodies: strategies and    procedures. Meth. Enzymol. 73, 3.-   Gupta, R. K., Varanelli, C. L., Griffin, P., Wallach, D. F. H.,    Siber, G. R. 1996. Adjuvant properties of non-phospholipid liposomes    (Novasomes®) in experimental animals for human vaccine antigens.    Vaccine 14, 219.-   Köhler, G., Milstein, C., 1975. Continuous cultures of fused cells    secreting antibody of predefined specificity. Nature 256, 495.-   Lohner, K., Degovics, G., Laggner, P., Gnamusch, E.,    Paltauf, F. 1993. Squalene promotes the formation of non-bilayer    structures in phospholipid model membranes. Biochim. Biophys. Acta    1152, 69.-   Koivisto, P. V. I., Miettinen, T. A. 1988. Increased amount of    cholesterol precursors in lipoproteins after ileal exclusion. Lipids    23, 993.-   Mayes, P. A. 1996. Cholesterol synthesis, transport, & excretion.    In: R. K. Murray, D. K. Granner, P. A. Mayes and V. W. Rodwell    (Eds.) Harper's Biochemistry, 24^(th) Edition, Appleton & Lang,    Stamsord, p. 271.-   Miettinen, T. A. 1982. Diurnal variation of cholesterol precursors    squalene and methyl sterols in human plasma lipoproteins. J. Lipid    Res. 23, 466.-   Minutello, M., Senatore, F., Cecchinelli, g., bianchi, M., Andreani,    t., Podda, A., Crovari, P. 1999. Safety and immunogenicity of an    inactivated subunit influenza virus vaccine combined with MF59    adjuvant emulsion in elderly subjects, immunized for three    consecutive influenza seasons. Vaccine 17, 99.-   Schuster, B., Neidig, M., Alving, B. M., Alving, C. R. 1979.    Production of antibodies against phosphocholine,    phosphatidylcholine, sphingomyelin, and lipid A by injection of    liposomes containing lipid A. J. Immunol. 122, 900.-   Stewart, M. E. 1992. Sebaceous gland lipids. Semin. Dermatol. 11,    100.-   Swartz, Jr., G. M., Gentry, M. K., Amende, L. M.,    Blanchette-Mackie, E. J., Alving, C. R. 1988. Antibodies to    cholesterol. Proc. Natl. Acad. Sci. U.S.A. 85, 1902.-   Stollar, B. D., McInerney, T., Gavron, T., Wassef, N. M., Swartz, G.    M., Jr., Alving, C. R. 1989. Cross-reactions of nucleic acids with    monoclonal antibodies to phosphatidylinositol phosphate and    cholesterol. Mol. Immunol. 26, 73.-   Wassef, N. M., Roerdink, F., Swartz, Jr., G. M., Lyon, J. A.,    Berson, B. J., Alving, C. R. 1984. Phosphate binding specificities    of monoclonal antibodies against phosphoinositides in liposomes.    Mol. Immunol. 21 863.

EXAMPLES Materials and Methods

Lipids

Squalene, squalane oils, and bovine serum albumin (essentially fattyacid free; cat. # A-7030) (BSA) were purchased from Sigma-AldrichChemical Company, St. Louis, Mo. Isopropanol was purchased from J. T.Baker, Phillipsburg, N.J. Emulsifiers for creating oil-in-wateremulsions consisted of Span 85 and Arlacel A (both from Sigma) and Tween80 (Aldrich Chemical Co., Milwaukee, Wis.). Dimyristoylphosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG),both used in the formation of liposomes, were purchased from AvantiPolar Lipids, Alabaster, Ala. Lipid A from Salmonella minnesota R595 waspurchased from List Biological Laboratories, Campbell, Calif. PVDFplates (Multiscreen-IP) were from Millipore, Bedford, Mass. Immullon 2 Uand flat bottom and Immulon 4HBX 96 well ELISA plates were from Dynex,Chantily, Va. F96 Maxisorp 96 well ELISA plates were from Nalge NuncInternational Corp., Naperville, Ill. Flat and U bottom tissue cultureplates were from Costar-Corning, Corning, N.Y. FBS was from GIBCO BRL,Grand Island N.Y. and was heated at 56° C. for 1 h prior to use. Gelatinwas from BioRad Laboratories, Richmond, Calif. Seal plate adhesive filmwas from PGC Scientific, Gaithersburg, Md. Affinity purified andadsorbed peroxidase-linked sheep anti-mouse IgM was from The BindingSite, San Diego, Calif. ABTS substrate was purchased from Kikegaard andPerry Laboratories, Gaithersburg, Md. Female Balb/c mice were purchasedfrom Jackson Laboratories, Bar Harbor, Me.

Immunologic and Culture Reagents

Aluminum hydroxide gel, Alhydrogel, was purchased from SuperfosBiosector, Vedbaek, Denmark. Mouse myeloma X63/Ag8.653 was purchasedfrom American Type Culture Collection, Chantilly, Va. Polyethyleneglycol 1500 was from Boehringer Mannheim, GmbH, Germany. Dulbecco'smodified Eagle's medium with high glucose (DMEM), MEM sodium pyruvate(100 mM), MEM nonessential amino acids (NEAA) (100×), penicillin (10,000units/ml)-streptomycin (10,000 μg/ml), 200 mM glutamine, 100×HAT (10 mMsodium hypoxanthine, 40 μM aminopterin, 1.6 mM thymidine) 100×HT (10 mMsodium hypoxanthine and 1.6 mM thymidine) supplements, Hank's BalancedSalts Solution, and fetal bovine serum were from GIBCO BRL, GrandIsland, N.Y. Fetal bovine serum was heated at 56° C. for 1 hour prior touse. Peroxidase-linked goat anti-mouse IgM and peroxidase-linked goatanti-mouse IgG were purchased from The Binding Site, San Diego, Calif.ATBS substrate was purchased from Kirkegaard & Perry Laboratories,Gaithersburg, Md. Gelatin was from BioRad Laboratories, Richmond, Calif.Polystyrene Immulon II ELISA plates “U” and flat bottom were from Dynex,Chantilly, Va. PVDF Multiscreen-IP plates were from Millipore Corp.,Bedford Mass. and adapted for ELISA. Seal plate adhesive film was fromPGC Scientific, Gaithersburg, Md. Sterile Dulbecco's phosphate bufferedsaline lacking calcium and magnesium (PBS) was from BioWhittaker,Walkersville, Md. Nonsterile PBS was prepared from standard laboratorysalts.

Manufacture of Liposomes

Liposomes containing SQE or SQA were prepared by a modification of themethod of Alving et al. (1993). DMPC and DMPG were dissolved inchloroform at 180 mM and 20 mM, respectively. Lipid A was dissolved inchloroform at a concentration of 1 mg/ml. Glassware was depyrogenatedovernight at 250° C. Chloroform solutions of lipids, including SQE orSQA, as appropriate, were placed in a pear shaped flask, and thechloroform was removed by rotary evaporation. The neck of the flask wascovered with sterile Whatman 541 filter paper to maintain sterility. Thedried lipid film was placed under high vacuum (50 mbar) for at least 1hr. PBS was added to the dried lipid film to give a final phospholipidconcentration of 100 mM. After closing with a ground glass stopper, theflask was shaken until all of the dried lipids were in suspension.Liposomes were stored at 4° C.

Liposomes Containing 43% Squalene for Immunization (Group 3)

Liposomes containing low amounts of SQE (43 mol %) were made withDMPC:DMPG:SQE in a molar ratio (9:1:7.5). Lipid A was added to give afinal dose of 25 μg in 0.2 ml of 100 mM phospholipid. Six ml of DMPC, 6ml of DMPG, 1.5 ml of lipid A, and 0.438 ml of SQE were added to a 100ml pear shaped flask. After drying as described above, PBS was added togive a final volume of 12 ml.

Liposomes Containing 71% Squalene for Immunization (Group 4)

Liposomes containing high amounts of SQE (71 mol %) were made withDMPC:DMPG:SQE in a molar ratio (9:1:25). Lipid A was added to give afinal dose of 25 μg in 0.2 ml of 100 mM liposomal phospholipid. Six mlof DMPC, 6 ml of DMPG, 1.5 ml of lipid A, and 1.46 ml of SQE were addedto a 100 ml pear shaped flask. After drying as described above, PBS wasadded to give a final volume of 12 ml.

Liposomes Used for ELISA

Liposomes used for ELISA were made with DMPC:DMPG or DMPC:DMPG:SQE (orSQA, as appropriate), in molar ratios of 9:1 or 9:1:7.5. Twenty ml ofDMPC, 20 ml of DMPG, and 1.44 ml of SQE or 1.6 ml of SQA (or no oilantigen) were added to a 100 ml pear shaped flask. After drying asabove, PBS was added and the final volume of the liposomes was adjustedto 20 ml. The liposomes are designated L(SQE) for SQE-containingliposomes, L(SQA) for SQA-containing liposomes, or L for liposomeslacking an oil antigen. The final phospholipid concentration was 100 mM.

Preparation of Emulsions for Immunization

Emulsion with 40% SQE, 10% Arlacel A, and Lipid A (Group 5)

Components for this formulation were initially prepared in two separate2 ml vaccine vials. One vial contained 1 ml of saline. For the secondvial, 2.5 mg of lyophilized lipid A was dissolved in 8 ml of SQE; 2 mlof Arlacel A were then added; and 1 ml of the combination was added tothe vial. The emulsion was prepared just prior to injection byemulsifying 0.75 ml of saline with 0.75 ml SQE-Arlacel A-lipid A using 2three ml plastic syringes and a 3-way stopcock. The saline was drawninto one syringe and the SQE-Arlacel A-lipid A was drawn into anothersyringe. The saline was pushed into the SQE-Arlacel A-lipid A. Themixture was passed back and forth at a rate of approximately 2passes/sec for 5 min. to form an emulsion. The emulsion was stable forseveral hours at room temperature.

Emulsion with 20% SQE, 5% Tween 80, 5% Span 85, and Lipid A (Group 6)

Components were vialed in two separate 2 ml vaccine vials prior toemulsification. One vial contained 1.5 ml of saline. The components forthe second vial were made by dissolving 12 mg of lyophilized lipid A in14.4 ml of SQE. Tween 80 (7.2 ml) and Span 85 (7.2 ml) were added to thelipid A in SQE. One ml of the mixture was vialed. The emulsion wasprepared just prior to injection by emulsifying 1.05 ml saline with 0.45ml SQE-Tween 80-Span 85-lipid A using 2 three ml plastic syringes and a3-way stopcock as described above. The emulsion was unstable andseparated into 2 layers in approximately 45 min.

Aluminum Hydroxide Gel Mixed with Emulsion Containing 19% Squalene, 1%Tween 80 and Lipid A (Group 7)

Aluminum hydroxide was diluted in saline to give 1.25 mg Al⁺³/ml and 1.5ml was placed in a 2 ml vaccine vial. The components for the second vialwere made by dissolving 4 mg of lyophilized lipid A in 6 ml of SQE.Tween 80 (0.32 ml) was added and 1.5 ml of the mixture was added to a 2ml vaccine vial. The formulation was prepared just prior to injection byemulsifying 1.2 ml of aluminum hydroxide in saline with 0.3 ml ofSQE-Tween 80-lipid A, as described above. The final aluminum hydroxideconcentration was 1 mg Al⁺³/ml. The mixture was unstable and separatedinto 2 layers in less than 30 min.

Aluminum Hydroxide Gel Mixed with Emulsion Containing 40% Squalene, 10%Arlacel A, and Lipid A (Group 8)

Aluminum hydroxide was diluted in saline to give 2 mg Al⁺³/ml, and 1.5ml was added to a 2 ml vaccine vial. The components for the second vialwere the same SQE-lipid A-Arlacel A mixture used in group 5. Theformulation was prepared just prior to injection by mixing 0.75 ml ofaluminum hydroxide in saline with 0.75 ml of SQE-Arlacel A-lipid A, asdescribed above. The final aluminum hydroxide was 1 mg Al⁺³/ml. Themixture was unstable and separated into 2 layers in less than 30 min.

TABLE I Summary of Immunization groups Group No. Antigen Composition* 1Squalene alone (0.5 ml) 2 Squalene (0.5 ml) mixed with 25 μg of lipid A3 Liposomes containing both lipid A and 43 mol % squalene 4 Liposomescontaining both lipid A and 71 mol % squalene 5 Emulsion containing 40%squalene, 10% Arlacel A, and lipid A 6 Emulsion containing 20% squalene,5% Tween 80, 5% Span 85, and lipid A 7 Aluminum hydroxide gel mixed withemulsion containing 19% squalene, 1% Tween 80 and lipid A 8 Aluminumhydroxide gel mixed with emulsion containing 40% squalene, 10% Arlacel Aand lipid A *All injections were administered i.p. in a 0.2 ml dose,except where indicated. Lipid A, when used, was administered at 25 μg oflipid A/dose.

Example 1 Immunizations

BALB/c mice, purchased from Jackson Labs. (Bar Harbor, Me.), wereimmunized i.p. and bled every 2 weeks under a protocol approved by theinstitutional Laboratory Animal Care and Use Committee. They were fedstandard mouse chow and water ad libitum. Groups of five mice receivedone of the following immunogens: Group 1—0.5 ml SQE; Group 2—0.5 ml ofSQE containing 25 μg lipid A; Group 3—0.2 ml of 43% SQE liposomes; Group4—0.2 ml of 71% SQE liposomes; Group 5—0.2 ml of emulsion containing 50%saline (0.9% sodium chloride), 40% SQE, 10% Arlacel A containing 25 μglipid A/dose; Group 6—0.2 ml of an emulsion containing 70% saline, 20%SQE, 5% Tween 80, 5% Span 85 (v/v) containing 25 μg lipid A/dose; Group7—0.2 ml aluminum hydroxide in saline, 19% SQE, 1% Tween 80, containing25 μg lipid A/dose; Group 8—0.2 ml of aluminum hydroxide in saline, 40%SQE, 10% Arlacel A containing 25 μg lipid A/dose (Table I). Animals wereboosted every 2 weeks. Three additional mice were immunized by theintravenous route with 0.2 ml of the high SQE liposomes (group 4). Threedays later, the animals were euthanized and the spleens removed forproduction of monoclonal antibodies.

Example 2 Production of Monoclonal Antibodies

Three days after the primary or boosting immunization, mice wereeuthanized and spleens obtained. Single cell suspensions of spleen cellswere prepared. Spleen cells and mouse myeloma X63/Ag8.653 cells werefused in a 1:1 ratio using polyethylene glycol 1500 (Köhler andMilstein, 1975; Galfré and Milstein, 1981). After fusion, the cells werecentrifuged and then suspended in DMEM containing 20% fetal bovineserum, 1 mM sodium pyruvate, 1×NEAA, 4 mM glutamine, 50 units/mlpenicillin, 50 μg/ml streptomycin, 1×HT (30 ml/spleen). Cells (0.1ml/well) were plated in 96 well plates. The next day 0.1 ml of DMEMmedia containing 1×HAT instead of HT was added to all of the wells. Ondays 2, 3, 5, 8, and 11, 0.1 ml of media was removed from each well and0.1 ml of DMEM containing HAT was added. After 8 days culturesupernatants were screened for antibodies reacting with SQE and not SQAby ELISA on PVDF plates as described below. Cells from culturesupernatants that were positive were expanded and then cloned twice bylimiting dilution.

Example 3 ELISA for Testing Serum for Antibodies to SQE UsingPolystyrene (PS) Plates

Solid-phase ELISAs were performed as described previously with minormodifications (Alving et al., 1996). For the initial serum screenassays, 10 μg of SQE or SQA in 50 μl of ethanol was placed in PS “U”bottom plates. The plates were placed overnight in a biological safetycabinet to allow the ethanol to evaporate. The plates were blocked with0.25 ml of PBS-0.3% gelatin for 2 h. After removal of the blockingbuffer, 50 μl/well of serum diluted in PBS-0.3% gelatin was added intriplicate. The plates were incubated at 4° C. overnight. The plateswere then washed 3 times with PBS using a plate washer (Skatron Inc.,Sterling, Va.). Peroxidase-labeled goat IgM (μ chain specific) werediluted 1000-fold in PBS-0.3% gelatin and 50 μl/well was added to theplates. Following incubation at room temperature for 1 h, the plateswere washed 3 times with PBS. ABTS substrate (50 μl/well) was added andthe plates were incubated for 1 h at room temperature in the dark. Theabsorbance at 405 nm was quantified using a UVmax Kinetic MicroplateReader (Molecular Devices, Palo Alto, Calif.). Assays were conducted intriplicate. Assay background was determined by incubation with wellslacking antigen. Background was subtracted from experimental values.Endpoint antibody titers were selected as the dilution at which theabsorbance was twice background.

Example 4 ELISA for Testing Culture Supernatants for Antibodies to SQEUsing PS Plates

For assay of culture supernatants of monoclonal antibodies, PS flatbottom plates were used. The assay was similar to that described abovefor the “U” bottom plates with the following changes. 1) The assayvolumes of coating antigen, primary and secondary antibodies andsubstrate was increased from 50 μl to 100 μl; 2) SQE and SQA weredissolved in isopropanol; 3) Incubation of with culture supernatants wasfor 1 h at room temperature instead of overnight at 4° C. These changesgave less background and somewhat greater reproducibility amongtriplicate determinations when compared to ELISA on PS “U” bottomplates. However, better results were obtained using PVDF membranes.

Example 5 ELISA for Antibodies to SQE Using PVDF Plates

The assay for antibodies to SQE was modified from the method describedfor detecting antibodies to cholesterol by Dijkstra et al. (1996). 0.1ml of SQE or SQA, as appropriate, dissolved in isopropanol were placedin each well and the plate was placed overnight in a biological safetycabinet to allow the isopropanol to evaporate. The wells were blockedwith PBS-4% FBS, pH 7.4, (0.3 ml/well) and incubated at room temperaturefor at least 1 hr. After removal of the blocking buffer, 0.1 ml ofculture supernatant (either undiluted or diluted in PBS-4% FBS) wasadded to each well. The plate was covered with seal plate adhesive filmand placed on a orbital shaker set at 1,500 rpm for 1 hr. The plateswere then washed 4 times with PBS-4% FBS. Sufficient PBS-4% FBS wasadded to each well until the air bubble floated off the PVDF membrane.Peroxidase-linked goat anti-mouse IgG or IgM was diluted 1 to 1000 inPBS-4% FBS and 0.1 ml was added to each well. The plates were coveredwith seal plate adhesive film and placed on the shaker as describedabove. The plates were washed 4 times with PBS as described above. ABTSsubstrate (0.15 ml/well) was added and the plates were covered with sealplate adhesive film. They were placed on the shaker, covered withaluminum foil, and shaken at 1,500 rpm. After 1 hr, 0.05 ml wastransferred from each well and placed in a corresponding well of 96 well“U” bottom plate. The absorbance was read at 405 nm using an ELISA platereader.

Example 6 ELISA Using L(SQE), L(SQA) and L as Capture Antigens

ELISAs using liposomes as capture antigens were performed using “U”bottom PS plates. L(SQE), L(SQA), or L, as appropriate, were diluted to660 nmol/ml in PBS (equivalent to 10 μg SQE). Fifty μl (33 nmol) wereplaced in each well. The plate was placed in a biological safety cabinetovernight. The plates containing the dried film of liposomes wereprocessed by ELISA as described in section 2.7. For serum assays, theplates containing diluted serum were incubated overnight at 4° C. Forassays using diluted supernatants from the monoclonal antibodies, theplates were incubated 1 h at room temperature.

Example 7 Induction and Reactivity of Polyclonal Antisera with SQE byELISA

Sera from immunized mice were tested by ELISA for the presence ofanti-SQE antibodies using SQE as the capture antigen. Among the eightimmunization strategies employed (see Materials and Methods, and summaryin Table I), only two groups exhibited increased IgM binding activityafter injection of the antigen when compared to the preimmunizationserum (group 4, FIG. 2A; group 6, FIG. 2B). None of the groups developedIgG binding activity after immunization (data not shown). Mice injectedwith liposomes containing lipid A and 71% SQE [L(71% SQE+LA)] (group 4,see Table I) showed progressively increased IgM titers with time whencompared to the pre-immunization bleeding (FIG. 2A). The animals wereimmunized every 2 weeks, and even at 2 weeks after a single injection,an increased IgM titer was evident. To a much lesser extent one of theSQE emulsion groups (group 6, see Table I) also developed increasedtiters when compared to the pre-immune sera, but even after multipleinjections there was no progressive increase in the antibody titer (FIG.2B). Because of this, in all further experiments sera from animalsimmunized with L(71% SQE+LA) was used.

Using an alternative capture antigen in the ELISA, namely liposomescontaining SQE an even higher resolution of positive results wasobserved when compared to the results obtained with SQE alone (FIG. 3A)as a capture antigen. However, as shown in FIG. 3, after immunizationwith liposomes containing SQE and lipid A, the antisera reacted not onlywith SQE alone but also with liposomes lacking SQE, albeit to a muchlesser extent than with liposomes containing SQE. This latterobservation is consistent with previous reports that antibodies tophospholipids are also induced when liposomal lipid A, or even lipid Aalone, is used as an adjuvant (Schuster et al., 1979; Banerji et al.,1981; Alving, 1986).

The above data suggested that antibodies that could react with SQE wereinduced in mice by immunization with certain formulations that containedSQE. However, when another oil molecule, SQA, the fully hydrogenatedform of SQE, was substituted for SQE as a capture antigen in the ELISA,the polyclonal antiserum to SQE reacted equally well with either SQE orSQA (data not shown). This apparent lack of monospecific binding to SQEcould have been due either to extensive cross-reactivity of anti-SQEantibodies with SQA, or to a mixed population of antibodies, some ofwhich cross-reacted with SQA and some of which did not. The possibilityof nonspecific binding of IgM antibodies also existed. Because of this,we decided to try to produce monoclonal antibodies that coulddifferentiate between SQE and SQA as antigens. In the course of thiswork, as shown below, we also refined the ELISA assay to minimizenonspecific effects and increase resolution.

Example 8 Development of Monoclonal Antibodies to SQE

To minimize experimental variation and nonspecific effects observedafter coating of hydrophobic antigens on polystyrene microtiter wells,we examined the possible benefits of coating the capture antigens onhydrophobic membranes consisting of polyvinylidene fluoride (PVDF), asdescribed by Aniagolu et al. (1995). As shown in FIGS. 4A and B, whenculture supernatants were assayed with PVDF membranes, an IgM anti-SQEmAb was identified that exhibited strong dose-dependent binding to SQE,but displayed little or no cross-reactivity to SQA. When the antigenswere coated on flat bottom PS microtiter wells instead of PVDFmembranes, the same anti-SQE mAb showed a complete lack of reactivitywith either SQE or SQA (FIGS. 4C and D).

Additional clones of anti-SQE mAbs were also produced which, when testedwith the PVDF membrane assay, either showed striking specificity for SQE(clone 5, FIG. 5), or reactivity with both SQE and SQA (clone 15, FIG.6). These data demonstrate that mAbs can be identified thatdifferentiate free SQE from free SQA by ELISA, particularly when theantigens are coated on PVDF membranes.

Example 9 Evaluation of the Specificity of mAbs for Reactivity with aCapture Antigen Consisting of Liposomes Containing SQE or SQA

The original immunizing antigen consisted of liposomes containingSQE+LA. FIG. 7 illustrates the results of ELISAs in which PS plates werecoated with liposomes containing or lacking SQE or SQA. An irrelevantIgM mAb (anti-asialoG_(M2)) is shown as a negative control (FIG. 7E).When analyzed for reactivity with liposomes containing SQE [L(SQE)],liposomes containing SQA [L(SQA)], or liposomes lacking both SQE and SQA[L], four different patterns of specificity for L(SQE), L(SQA), and Lalone were observed, as derived from FIG. 7 and summarized in Table II.It is noteworthy that we have never obtained a mAb that bound morestrongly to SQA than to SQE. This is in keeping with the primaryspecificity of the antibodies for liposomal SQE.

Evaluation of the Specificity of Polyclonal Antiserum for SQE and SQA onPVDF Membranes.

The above studies demonstrate that immunization with SQE induces a mixedpopulation of anti-SQE antibodies that includes some that do notcross-react and some that do cross-react with SQA. In view of this,polyclonal anti-SQE antiserum would be expected to exhibit both SQEreactivity and SQA cross-reactivity on PVDF membranes. As shown in FIG.8, reactivity with both antigens was observed with polyclonal anti-SQEantiserum.

To demonstrate that specific antibodies to SQE actually do exist, wecreated mAbs that were selected for the ability to bind to SQE but had arelative inability to bind to SQA, as determined by ELISA withhydrophobic PVDF membranes. Monoclonal antibodies were successfullycreated that specifically bound to SQE but to a lesser extent, or not atall, to SQA. However, numerous anti-SQE mAbs were also created thatcross-reacted strongly with SQA. It is concluded that specificdifferentiation of SQE from SQA demonstrates that the unsaturated bondsof SQE can play a major role in the specificity of the antibodies, andsuch antibodies therefore have a distinctive conformational specificity.However, the extensive cross-reactivity of numerous clones of anti-SQEantibodies with SQA also demonstrates that the unsaturated bonds are notthe sole determinant of specificity.

Example 10

We have demonstrated in this study that polyclonal and monoclonalantibodies that bind to SQE can be developed after immunization of micewith liposomes containing 71% SQE and lipid A. Other methods ofimmunization, including immunizing with liposomes containing 43% SQE orwith a variety of SQE-containing emulsions, were either completelyineffective, or considerably less effective, as immunogens. The strategyof utilizing liposomes containing 71% SQE and lipid A as an immunogenwas modeled after similar success in the induction of antibodies tocholesterol by immunizing with liposomes containing 71% cholesterol andlipid A (Swartz et al., 1988; Alving and Swartz, 1991; Dijkstra et al.,1996). Although we have previously found that simple injection ofsilicone oil into mice can also cause the induction of antibodies tocholesterol (Alving et al., 1996), injection of non-emulsified SQE oilmixed with lipid A did not result in the induction of antibodies to SQE(group 2, Table I). Among four emulsions containing SQE and lipid A ascomponents, only one (group 6, Table I) induced any immune response toSQE, and this was quite weak even after multiple injections (FIG. 2B).From these data we conclude that SQE is a very poor antigen when usedeither as an oil or an emulsion, even when lipid A, a potent adjuvantfor inducing antibodies to lipids, is included in the immunizingformulation.

The results in the present study are consistent with the concept ofinduction of anti-liposome mAb antibodies having specificities thatinclude both liposomal phospholipid as well as SQE in the antigenbinding site of the antibody (FIGS. 7B and C; Table II). However, aswith the anti-cholesterol mAbs, anti-SQE clones were also obtained thatdid not react with liposomal phospholipid (FIGS. 7A and D; Table II).

TABLE II Monoclonal Antibody Specificities Obtained After Injection ofLiposomes Containing Lipid A and SQE Binding Specificity* Clone No. SQESQA Liposomal phospholipid 15 + − − 4 + + + 18 + − + 14 + + − *Based ondata from Figure 7.

Example 11

The extreme hydrophobicity of SQE raises an important theoreticalproblem in demonstrating specificity of antibodies because polyclonalantiserum raised by immunization with SQE shows considerable reactivitywith SQA (FIG. 8). Based on serum data alone, it was therefore initiallyimpossible to determine whether the apparent antibody activity in theantiserum is specific to SQE, or if the immunoglobulins are simplynonspecifically binding hydrophobically both to SQE and SQA. Our initialexperiments using PS microtiter plates did indeed demonstratenonspecific hydrophobic binding of IgM molecules to both SQE and SQA,and other alkanes (data not shown). However, this problem was solved bycoating the antigens on hydrophobic PVDF membranes, as described byAniagolu et al. (1995). Although commercially-available PVDF membranesalso present the problem that they are physically located in PSmicrotiter wells, they apparently do have the salutary effect ofblocking most or all of the nonspecific hydrophobic binding sites of thealkane molecules.

Example 12

Culture supernatants containing monoclonal antibodies (mAbs) to SQE weregrown in Dubbelco's modified Eagle's medium as described (Matyas) Micewere injected with liposomes containing 71 mol percent SQE and lipid A,intrapertioneally as described (matyas). Anti-SQE positive serum wasobtained by terminal bleeding three days after immunization. The serumwas aliquoted and frozen at −20° C. The experiments described in thispaper used several different lots of monoclonal supernatants andanti-SQE serum.

Example 13 ELISA Assay on PVDF Plates

The ELISA assay in plates containing PVDF membranes were conducted asdescribed (matyas). Briefly, SQE was diluted in isopropanol and placedin the wells of the plate. After drying overnight, the wells wereblocked with PBS-4% FBS, pH 7.4, for 2 h. Serum and supernatantscontaining monoclonal antibodies to SQE were diluted in PBS-4% FBS and0.1 ml was placed in a well. Following incubation for 1 h, the plate waswashed four times with PBS-4% FBS by hand using a 25 ml pipet. 0.1 ml ofperoxidase-linked sheep anti-mouse IgM diluted 1:1000 in PBS-4% FBS wasadded to each well. The plate was incubated for 1 h and then washed 4times with PBS by hand. 0.15 ml of ABTS substrate was added to each welland the plates were incubated for 1 h. 0.1 ml/well was transferred to anImmulon 2 U bottom plate and the absorbance was read at 405 nm with aUvmax Kinetic Microplate Reader (Molecular Devices, Palo Alto, Calif.).In some experiments PVDF plates were washed 4 times with 0.5 ml ofPBS/well with an ELISA plate washer (Skatron, Sterling, Va.). The vacuumprobes of the washer were positioned just above the PVDF membrane.

Example 14 ELISA Assay on Polystyrene Plates

The ELISA assay for polystyrene plates was performed as described(Matyas). The assay for polystryrene tissue culture U bottom plates isdescribed in detail. SQE was diluted in isopropanol (1 μmol/ml; 24 μlSQE/50 ml) and 0.1 ml was placed in each well. Control wells wereisopropanol alone. The plates were placed in a biological safety cabinetand incubated overnight to allow the isopropanol to evaporate. PBS-4%FBS, pH 7.4 was added to each well (0.3 ml/well) to block unboundbinding sites. After incubation at room temperature for 2 h, the plateswere dumped and tapped on paper towel to removed the blocked. Culturesupernatants containing monoclonal antibodies to SQE or mouse serum wasdiluted in PBS-2% BSA and added to the plates in triplicate. Followingincubation for 1 h at room temperature, the plates were washed 4 timeswith 0.5 ml of PBS/well using a Skatron plate washer. Peroxidase-linkedsheep anti-mouse IgM was diluted 1:1000 in PBS-4% FBS and 0.1 ml wasadded to each well of the plate. The plates were incubated 1 h at roomtemperature and washed 4 times with PBS. ABTS substrate (0.1 ml/well)and the plates were incubated at room temperature for 1 h. Absorbancewas read at 405 nm.

Example 15 Effect of Polystyrene Plate Type on the Measurement ofAntibodies to SQE

Seven different plates coated with SQE as an antigen were compared fortheir ability to detect monoclonal antibodies and serum antibodies toSQE. Hand washed Millipore IP plates had low background(isopropanol-treated wells) and high anti-SQE absorbances for the mAbs,but low absorbances to SQE-coated wells were obtained for anti-SQE serum(FIG. 9A). When the Millipore IP plates were washed with an ELISA platewasher, the background absorbances for the anti-SQE serum significantlyincreased (FIG. 9B). Immulon 2 U and flat bottom plates had very highbackground absorbances for the mAbs and the anti-SQE serum (FIGS. 9G,H). Immulon 4HBX had elevated absorbances for SQE-coated wells that werenot incubated with primary antibody (FIG. 9E). Maxisorp F96 plates hadelevated background absorbances with anti-SQE serum. Costar U andflat-bottom tissue culture plates had low background absorbances andhigh absorbances for SQE-coated wells with both the mAbs and anti-SQEserum (FIGS. 9C, D). There were no real differences in ELISA valuesobserved between the U bottom and flat bottom Costar tissue cultureplates. The Costar U bottom plate was chosen for the assay of antibodiesto SQE.

Example 16 Standardization of the ELISA Assay for Anti-SQE Antibodies onCostar U Bottom Tissue Culture Plates

The plates were tested with various blocker/diluents in order tominimize background and maximize antibody binding to SQE. PBS-0.5%casein effectively abolished the binding of the mAbs to SQE (FIGS. 10A,B) and elevated the background with the anti-SQE serum (FIG. 11D).PBS-0.3% gelatin and PBS-0.6% gelatin inhibited the binding of the mAbsto SQE-coated wells (FIGS. 10A, B). In addition, when gelatin was usedas a blocker/diluent, the results from experiment to experiment werehighly variable. When PBS-1% BSA was used as a blocker/diluent,absorbances increased over 2-fold for the mAbs on SQE-coated wellscompared to similar wells using PBS-4% FBS as a blocker/diluent (FIGS.10A, B). Background absorbances with PBS-1% BSA were similarly increasedapproximately 2-fold for both the mAbs, normal mouse and anti-SQE serum(FIG. 10). Various concentration of BSA were tested to determine if thebackground could be reduced. mABs and anti-SQE serum had highabsorbances for SQE-coated wells using PBS-1% and 2% BSA asblockers/dilutents (FIG. 11). BSA concentrations greater than 2% causedsignificant reductions in the absorbances of SQE #18 (FIG. 11B) andanti-SQE serum (FIG. 11D). Background absorbances forisopropanol-treated wells were greatly reduced by increasing the BSAfrom 1% to 2%, but did were not dramatically reduce further withincreasing concentrations of BSA (FIG. 11). PBS-2% BSA was chosen as theblocker/diluent.

The optimal incubation temperature was investigated. The binding ofclone SQE #16 was independent of the incubation temperature (FIG. 12A).Maximal binding of anti-SQE serum to SQE-coated wells occurred attemperatures above 4° C. (FIG. 12B). Background binding of anti-SQEserum to isopropanol-treated wells was relatively independent oftemperature. There was a slight increase in binding toisopropanol-treated wells at 22° C. Since binding was basicallyindependent of temperature, room temperature was chosen for the standardassay.

A comparison of the time required for primary antibody binding toSQE-coated wells indicated that overnight incubation did not increasethe binding of clone SQE #16 to SQE-coated wells (FIG. 13A). There wasincreased binding of anti-SQE serum to SQE-coated wells with overnightincubation, but this was mostly offset by and an increase in backgroundbinding to isopropanol-treated wells (FIG. 13B). One hr was chosen asthe incubation time for primary antibody. Similarly, there was nodifference between incubating with secondary antibody for 1 or 2 hr forboth clone SQE #16 and anti-SQE serum (FIG. 14). Consequently, 1 hr waschosen as the incubation for secondary antibody.

The binding of mAbs SQE #14 and SQE #16 was dependent upon the amount ofSQE added to the wells (FIG. 15A, B). Maximal binding was observed from10 to 25 nmol of SQE. Maximal absorbances for anti-SQE serum wereobtained from 7.5 to 100 nmol of SQE. SQE amounts above or below thoseamounts had decreased absorbances. Control mouse IgM monoclonal antibodydid not bind to SQE at any amount coated on the plate (FIG. 15A). Tennmol of SQE was chosen as the preferred amount for the standard assay.

Example 17 Conditions for the Standard Assay for Measuring Antibodies toSQE

Based on the results described above, the following conditions wereadopted as the standard assay conditions:

1. Costar 96 well “U” bottom sterile tissue culture plates were chosen.

2. A SQE concentration of 10 nmol SQE/well in 0.1 ml of isopropanol waschosen. The isopropanol was allowed to evaporate over night in abiological safety cabinet with the air turned-on.

3. The plates were blocked with 0.3 ml/well of PBS-2% BSA, pH 7.4 for 2hr at room temperature.

4. Mouse serum or monoclonal antibodies are diluted in PBS-2% BSA.

5. The plates are dumped and tapped on paper towels. 0.1 ml/well ofdiluted serum or monoclonal antibody was added to the plate. The plateswere covered and incubated at room temperature for 1 hr.

6. The plates were washed 4 times with PBS, pH 7.4 with 0.5 ml/well.

7. Peroxidase-linked sheep anti-mouse IgM was diluted 1:1000 in PBS-2%BSA and 0.1 ml was added to each well. The plates are covered andincubated at room temperature for 1 hr.

8. The plates were washed 4 times with PBS, pH 7.4 at 0.5 ml/well.

9. 0.1 ml of ABTS substrate was added to each well. The plates werecovered with foil and incubated at room temperature for 1 hr.

10. The absorbance was read at 405 nm.

Example 18 Reproducibility of the Assay

Several different lots of Costar U bottom tissue culture plates weretested under the standard assay conditions. There was no differenceamong the absorbances obtained with the mAbs on the different lots ofplates (FIGS. 16A, B). Slight differences in absorbances were observedwith different lots of plates with anti-SQE serum on SQE-coated wells(FIG. 17C). Background absorbances were the same on the different lotsof plates for both the monoclonal antibodies and the anti-SQE serum. Theassay was highly reproducible from day to day both with the monoclonalantibodies (FIG. 17A) and with anti-SQE serum (FIG. 17B).

Example 19 Detection of Antibodies to Squalene in Human Sera

Squalene and squalane oils were purchased from Sigma-Aldrich ChemicalCompany, St. Louis, Mo. Isopropanol and casein were purchased from J. T.Baker, Phillipsburg, N.J. Gelatin was from BioRad Laboratories,Richmond, Calif. Flat and U bottom tissue culture plates were fromCostar-Corning, Corning, N.Y. Affinity purified and adsorbedperoxidase-linked sheep anti-human IgG and IgM was from The BindingSite, San Diego, Calif. ABTS substrate was purchased from Kirkegaard andPerry Laboratories, Gaithersburg, Md. Human serum samples were obtainedunder IRB approved protocol from Phillip Pittman at the United StatesArmy Medical Research Institute of Infectious Disease, Frederick, Md.

ELISA Assay

Squalene was diluted in isopropanol to 0.2 μmol/ml (9.6 μl squalene/100ml) and 0.1 ml was placed in each well. Control wells containedisopropanol alone. The plates were placed in a biological safety cabinetand incubated overnight to allow the isopropanol to evaporate. PBS-0.5%casein, pH 7.4, was added to each well (0.3 ml/well). After incubationat room temperature for 2 h, the plates were dumped and tapped on apaper towel to removed the blocking buffer. Serum samples were dilutedin PBS-0.5% casein and added to the plates in triplicate. Followingovernight incubation at room temperature, the plates were washed 4 timeswith 0.5 ml of PBS/well using a MAP-C ELISA workstation (Titertek,Huntsville, Ala.). Peroxidase-linked sheep anti-human IgG and IgM wasdiluted 1:1000 in PBS-0.5% casein and 0.1 ml was added to each well ofthe plate. The plates were incubated 1 h at room temperature and washed4 times with PBS. ABTS substrate (0.1 ml/well) and the plates wereincubated at room temperature for 1 h. Absorbance was read at 405 nm.

Serum Anti-Squalene Grading Criteria

POSITIVE—The absorbance for the squalene-coated wells was at least 2.5times that of isopropanol-treated wells and 10 times the absorbance forsqualene-coated wells that were not incubated with primary antibody.

INCONCLUSIVE—(high background) The absorbance for isopropanol-treatedwells was at least 5 times the absorbance of isopropanol-treated wellsthat were not incubated with primary antibody and the absorbance for thesqualene-coated wells was at least 10 times the absorbance forsqualene-coated wells that were not incubated with primary antibody.

NEGATIVE—The absorbances failed to meet the above criteria.

Summary

A. 4.1% of the serum samples (8 of 197) were positive for IgG antibodiesto squalene.

B. 95.9% of the serum samples (189 of 197) were negative for IgGantibodies to squalene.

C. 10.2% of the serum samples (20 of 197) were positive for IgMantibodies to squalene.

D. 14.2% of the serum samples (28 of 197) were inconclusive for IgMantibodies to squalene. These sera had high background binding toisopropanol-treated wells.

E. 75.6% of the serum samples (149 of 197) were negative for IgMantibodies to squalene.

F. Most of the positive anti-squalene samples had endpoint titers of100-200.

The results for these experiments are shown graphically in FIGS. 18-22.

Although the present invention has been described in terms of aparticular preferred embodiments, it is not limited to thoseembodiments. Alternative embodiments, examples, and modifications whichwould still be encompassed by the invention may be made by those skilledin the art, particularly in light of the foregoing teachings.

1. An assay method for detecting the presence of squalene antibodiescomprising the steps of providing a solid support immobilized withsqualene thereon, adding bovine serum albumin as a blocking agent tosaid solid support, and contacting the immobilized squalene with a serumsample containing squalene antibodies to form an antibody complex, andbinding the antibody complex with a labeled ligand for detecting thepresence of squalene antibodies in said serum sample.
 2. The method ofclaim 1 wherein the method further comprises using a monoclonal antibodyto specifically bind to any squalene antibodies present in the serum. 3.A method for detecting squalene antibodies in serum comprising the stepsof immobilizing squalene on a solid support, contacting the immobilizedsqualene with a serum sample containing squalene antibodies to form anantibody complex, and binding the antibody complex with a ligand inwhich the ligand is an antibody capable of capturing said antibodycomplex.
 4. A method for detecting the presence of squalene antibodiescapable of specific binding with squalene, comprising: providing a solidsupport suitable for allowing specific binding of squalene with squaleneantibodies; immobilizing squalene on the solid support; allowingsqualene antibodies to specifically bind to the immobilized squalene toform a specific antibody complex; contacting the antibody complex withan indicator agent specifically binds to the complex; and detecting theindicator agent.
 5. The method of claim 4 further comprising the step ofblocking the immobilized squalene with a blocking agent to reducebackground interference with antibody binding to the immobilizedsqualene.
 6. The method of claim 4 where the detection is part of anELISA protocol.